Single-molecule analysis of transcription factor binding at transcription sites in live cells

Abstract

Although numerous live-cell measurements have shown that transcription factors (TFs) bind chromatin transiently, no measurements of transient binding have been reported at the endogenous response elements (REs) where transcription is normally induced. Here we show that at endogenous REs the transcriptionally productive specific binding of two TFs, p53 and the glucocorticoid receptor (GR), is transient. We also find that the transient residence times of GR at endogenous REs are roughly comparable to those at an artificial, multi-copy array of gene regulatory sites, supporting the use of multi-copy arrays for live-cell analysis of transcription. Finally, we find that at any moment only a small fraction of TF molecules are engaged in transcriptionally productive binding at endogenous REs. The small fraction of bound factors provides one explanation for gene bursting and it also indicates that REs may often be unoccupied, resulting in partial responses to transcriptional signals.

Figure 1

Images of GR single molecules in the nucleus of a live cell. HaloTMR-tagged GR molecules at selected time points (upper left corner) are presented from a 60 s movie. Projection of 85 successive timepoints from the movie defines the boundary of the nucleus (first frame). Color coding in subsequent panels indicates three separate segments of the movie illustrating a bound molecule (yellow - residence time of 10 s), a molecule that appears for only one timepoint (green) and a free molecule that diffuses in the plane of focus (red). Arrows indicate the molecule of interest. Red dotted line indicates the path of the diffusing molecule. Scale bar 1 µm.

Figure 2

Single molecule tracking within and outside of transcription domains. Fixed cells showing GFP-tagged polymerase II puncta (a) with BrUTP incorporation marking sites of transcription (b). Many polymerase II puncta are sites of BrUTP incorporation (overlay, c). Four time points (shown in upper left corner) of a GFP-polymerase II movie taken in live cells (d), and the projection image from that movie (e). Yellow arrows indicate examples of polymerase II foci that appear in multiple frames and give rise to well-defined domains in the averaged movie. Red arrow indicates a region largely devoid of polymerase. Dotted circles (0.6 µm diameter) provide examples of regions where single molecule tracking would be performed. (f) Selected time points from a movie showing association of a single GR molecule (red) with an averaged projection of a polymerase II movie (green). White arrows indicate the frames (0.4 s – 8.0 s) during which the GR molecule remains bound within the transcription domain. (g) Selected time points from a movie showing a bound GR molecule away from a transcription domain, with binding occurring from 0.2 – 2.0 s. Scale bars 1 µm.

Figure 3

Cumulative histograms with semi-log plots (insets). Dwell times of molecules in transcription domains are significantly longer than dwell times of molecules away from transcription domains for both p53 (a) and GR (b). The tail of the histogram for the seven-amino-acid specific-site binding mutant p53 mSB disappears, while a reduction in the height of the tail is seen for the single-amino-acid mutant p53 R273H (c).The tail of the historgram of GR activated with corticosterone fell to zero earlier compared to dexamethasone activation (d). We collected 20 – 30 time-lapse movies for each experimental condition. From this set of movies we selected 2 – 3 representative movies in which the number and brightness of the single molecules were optimal for tracking, and then from these movies we tracked a total of 400 – 700 single molecules for each condition. See Fig. S6 for histograms of p53 mSB, p53 R273H and GR outside of transcription domains, and Fig. S5 for representative fits to these data.

Figure 4

GR binding at the MMTV tandem array. The MMTV tandem array (bright green puncta in a) is typically associated with a distinct cluster of polymerase II puncta (b,c). This cluster of polymerase II at the array can also be discerned in live cells (white box in d), and used to define a transcriptionally active zone at the array (yellow outline in the inset). (e) A much higher fraction of molecules with longer dwell times are found at the array compared to randomly selected transcription sites. Scale bars 1 µm.